John Carroll University Carroll Collected Masters Theses Theses, Essays, and Senior Honors Projects Summer 2016 SEQUESTERED ALKALOID DEFENSES IN THE DENDROBATID POISON FROG OOPHAGA PUMILIO PROVIDE VARIABLE PROTECTION FROM MICROBIAL PATHOGENS Kyle Hovey John Carroll University, [email protected] Follow this and additional works at: http://collected.jcu.edu/masterstheses Part of the Biology Commons Recommended Citation Hovey, Kyle, "SEQUESTERED ALKALOID DEFENSES IN THE DENDROBATID POISON FROG OOPHAGA PUMILIO PROVIDE VARIABLE PROTECTION FROM MICROBIAL PATHOGENS" (2016). Masters Theses. 19. http://collected.jcu.edu/masterstheses/19 This Thesis is brought to you for free and open access by the Theses, Essays, and Senior Honors Projects at Carroll Collected. It has been accepted for inclusion in Masters Theses by an authorized administrator of Carroll Collected. For more information, please contact [email protected]. SEQUESTERED ALKALOID DEFENSES IN THE DENDROBATID POISON FROG OOPHAGA PUMILIO PROVIDE VARIABLE PROTECTION FROM MICROBIAL PATHOGENS A Thesis Submitted to the Office of Graduate Studies College of Arts & Sciences of John Carroll University in Partial Fulfillment of the Requirements for the Degree of Master of Science By Kyle J. Hovey 2016 Table of Contents Abstract ................................................................................................................................1 Introduction ..........................................................................................................................3 Methods Study Sites and Frog Collections ...........................................................................12 Alkaloid Fractionations ..........................................................................................14 Culture and Maintenance of Microbes ...................................................................15 Microbial Growth Inhibition Assays .....................................................................16 Alkaloid Analysis...................................................................................................19 Statistical Analysis .................................................................................................20 Results Alkaloid Analysis...................................................................................................21 Dose-Response Relationship between 2.4 µL and 4.8 µL Treatments ..................22 Aeromonas hydrophila Optical Density Assays ....................................................22 Aeromonas hydrophila Colony-Forming Unit Assays ..........................................23 Klebsiella pneumoniae Optical Density Assays ....................................................24 Klebsiella pneumoniae Colony-Forming Unit Assays ..........................................24 Relationships between Alkaloid Composition and Growth Inhibition ..................25 Relationship between Optical Density and Colony-Forming Unit Assays ............26 Discussion ..........................................................................................................................26 Acknowledgments..............................................................................................................44 Literature Cited ..................................................................................................................46 Tables and Figures Table 1 ...................................................................................................................74 Table 2 ...................................................................................................................75 Table 3 ...................................................................................................................76 Table 4 ...................................................................................................................77 Figure 1 ..................................................................................................................78 Figure 2 ..................................................................................................................79 Figure 3 ..................................................................................................................80 Figure 4 ..................................................................................................................81 Figure 5 ..................................................................................................................82 Figure 6 ..................................................................................................................83 Figure 7 ..................................................................................................................84 Figure 8 ..................................................................................................................85 Appendices Cryopreservation of Microbes ...............................................................................86 Pilot Experiments ...................................................................................................86 Aeromonas hydrophila OD Pilot Assays ...............................................................86 Aeromonas hydrophila CFU Pilot Assays .............................................................92 Klebsiella pneumoniae OD Pilot Assays ...............................................................92 Klebsiella pneumoniae CFU Pilot Assays .............................................................93 Batrachochytrium dendrobatidis (Chytrid Fungus) Culture and Pilot Assays ......94 Appendix 1 .............................................................................................................99 Appendix 2 ...........................................................................................................100 Appendix 3 ...........................................................................................................100 Appendix 4 ...........................................................................................................101 Appendix 5 ...........................................................................................................102 Appendix 6 ...........................................................................................................103 Appendix 7 ...........................................................................................................104 Appendix 8 ...........................................................................................................105 Appendix 9 ...........................................................................................................106 Appendix 10 .........................................................................................................107 Appendix 11 .........................................................................................................108 Appendix 12 .........................................................................................................108 Appendix 13 .........................................................................................................109 Appendix 14 .........................................................................................................109 Appendix 15 .........................................................................................................110 Appendix 16 .........................................................................................................110 Appendix 17 .........................................................................................................111 Appendix 18 .........................................................................................................111 Appendix 19 .........................................................................................................112 Appendix 20 .........................................................................................................112 Appendix 21 .........................................................................................................113 Appendix 22 .........................................................................................................113 Appendix 23 .........................................................................................................114 Appendix 24 .........................................................................................................114 Appendix 25 .........................................................................................................115 Appendix 26 .........................................................................................................115 Appendix 27 .........................................................................................................116 Appendix 28 .........................................................................................................116 Appendix 29 .........................................................................................................117 Appendix 30 .........................................................................................................117 Appendix 31 .........................................................................................................118 SEQUESTERED ALKALOID DEFENSES IN THE DENDROBATID POISON FROG OOPHAGA PUMILIO PROVIDE VARIABLE PROTECTION FROM MICROBIAL PATHOGENS ABSTRACT Amphibians produce the majority